Karnovsky’s fixative prevents artifacts appearing as vacuolation derived from tissue processing in kidneys treated with antisense oligonucleotide

Antisense oligonucleotide (ASO) therapies have been identified as a new treatment modality for intractable diseases. In kidneys treated with ASOs, vacuoles, in addition to basophilic granules, are often observed in the proximal tubules. Some reports have described that these vacuoles are likely to be a secondary phenomenon resulting from the extraction of ASOs during tissue processing. In this study, we compared renal morphology after fixation with Karnovsky’s fixative or 4% paraformaldehyde phosphate buffer (PFA) with that of 10% neutral-buffered formaldehyde solution (NBF). Female Sprague-Dawley rats, intravenously treated four times with 50 mg/kg locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) for 1 or 2 weeks, were examined. Microscopically, vacuoles and basophilic granules in the proximal tubules were observed in the kidneys fixed with NBF. Basophilic granules are indicative of the accumulation of ASOs. Moreover, some of the vacuoles also contained faint basophilic granules, suggesting that the vacuoles were relevant to the accumulation of ASOs. Although moderate vacuolation was observed in the proximal tubules, the majority of the vacuolated epithelia were negative for kidney injury molecule-1 on immunohistochemical staining. Vacuoles in the proximal tubules were not observed in samples subjected to Karnovsky’s fixation, although basophilic granules were observed. In samples subjected to PFA fixation, vacuoles and basophilic granules were observed in the proximal tubules, similar to those in samples subjected to NBF fixation. Overall, our findings demonstrated the possibility of overestimation of vacuolation due to artifacts during tissue processing when using conventional NBF fixation. Karnovsky’s fixative is considered a useful alternative for distinguishing artificial vacuoles from true nephrotoxicity.     

Ultrastructural alterations in the kidneys of Pekin ducks fed methylmercury.

Ultrastructural alterations in the kidneys of Pekin ducks were studied after subchronic exposure to methylmercury chloride. Twenty-four male and twenty-four female Pekin ducks, 12 weeks of age were fed 0.0 (control), 0.5, 5.0 and 15.0 parts per million of MeHgCl, mixed in a mash diet for 12 weeks. Birds were observed for clinical signs daily. Kidneys were fixed in situ by perfusion with 4% glutaraldehyde in 0.05 M phosphate buffer and routinely processed for electron microscopy. Only minor morphological changes were observed in the renal corpuscle of ducks in the 0.5 ppm group. The component cells of the renal corpuscle in the 5.0 and 15.0 ppm groups showed several ultrastructural alterations, including an increase in number and size of lysosomes and vacuoles in podocytes and mild vacuolation in mesangial cells. The juxtaglomerular cells of afferent arterioles had crystallized granules. No significant changes were observed in glomerular basement membrane thickness and width of podocyte pedicels. The proximal tubules of the 5.0 and 15.0 ppm groups showed some microvillar loss and an increase in the number and size of lipid droplets, vacuoles and lysosomal bodies. The distal tubular changes in the 5.0 and 15 ppm groups included electron lucency and vacuolation of cytoplasm as well as occasional mitochondrial swelling. The collecting ducts of 5.0 ppm and 15.0 ppm groups showed varying degrees of degenerative changes in both mucus secretory as well as absorptive cells. These results suggest that subchronic exposure of Pekin ducks to methylmercury chloride causes subtle ultrastructural changes in renal corpuscle and marked ultrastructural changes in proximal tubules and collecting ducts.     

Ochratoxin A and citrinin induced nephrosis in Beagle dogs. III. Terminal renal ultrastructural alterations

The extent and type of renal ultrastructural changes in Beagle dogs varied with the administration of ochratoxin A and citrinin alone and in the two dosage combinations. The three predominant changes were cytoplasmic vacuolation, myelin figure formation and lesions designated as cytoplasmic disarray. These changes were mainly of the endomembane system of the tubular epithelial cells. Cytoplasmic vacuoles were within proximal and distal tubules and collecting ducts and were most numerous in dogs given 10 mg/kg critrinin. Vacuolation of similar distribution, but less severe, was seen in renal tubular cells of dogs given the higher dose of the combined mycotoxins (0.2 mg/kg ochratoxin A + 10 mg/kg citrinin). This damage was limited to the proximal tubular cells in dogs given only ochratoxin A (0.1 or 0.2 mg/kg). Myelin figures were in proximal epithelial cells of dogs given ochratoxin A alone or combined with citrinin. There was cytoplasmic disarray in dogs of all groups except for dogs given 5 mg/kg citrinin. This lesions was usually limited to the proximal tubules. The lesions, however, was found in cells of the distal tubules of dogs given 10 mg/kg citrinin alone.     

Renal lesions in spontaneous insulin-dependent diabetes mellitus in the nonobese diabetic mouse: acute phase of diabetes

The nonobese diabetic mouse is a model of spontaneous insulin-dependent diabetes mellitus. The present study made longitudinal observations of renal lesions in the acute-progressive phase of diabetic mice 0, 10, 20, 30, and 40 days after onset of diabetes without insulin therapy. Plasma creatinine and blood urea nitrogen concentrations gradually increased after onset of diabetes. Kidney weight increased and plateaued at day 20. Under electron microscopy the glomeruli demonstrated only mild changes on day 40. In the proximal tubules proliferating cell nuclear antigen-positive nuclei and nuclear divisions were increased on days 10 and 20. On day 40 of diabetes, increased periodic acid-Schiff-positive granules, confirmed as lysosomal dense bodies, increased neuronal nitric oxide synthase (nNOS) positive reaction, and decreased periodic acid-Schiff staining in the brush border were observed in the proximal straight tubules. In the juxtaglomerular apparatus stratified macula densa were decreased with time in diabetes compared with the findings on day 0, and this macula densa positively reacted with nNOS. No changes in renin levels were observed. In addition, apoptotic cells were not detected. In conclusion, this research represents the first thorough characterization of acute changes in nonobese diabetic mouse kidneys. The results demonstrated renal hypertrophy and slight glomerular injury in early stages and structural alteration of the proximal straight tubules at later stages during the acute phase of diabetes. Furthermore, increased nNOS may represent one of the pathogenic factors of diabetic nephropathy.     

Spontaneous and experimental glycoprotein storage disease of goats induced by Ipomoea carnea subsp fistulosa (Convolvulaceae)

Spontaneous and experimental poisoning with the swainsonine-containing and calystegine-containing plant Ipomoea carnea subsp fistulosa is described. Three of 8 goats presenting with emaciation, weakness, symmetrical ataxia, posterior paresis, proprioceptive deficits, abnormal posture, abnormal postural reaction, and muscle hypertonia were necropsied. I fistulosa was suspected to be the cause of the neurologic disease in all cases. An experiment was conducted to confirm the diagnosis using 12 goats and diets containing 3 different concentrations of the plant. All goats fed I fistulosa developed neurological signs that were similar to those observed in the spontaneous intoxication. Muscle atrophy and pallor were the only macroscopic changes observed in spontaneous and in experimental intoxication. Histological lesions of spontaneous and experimental animals were similar. The most prominent lesion was cytoplasmic vacuolation in neurons of the central and the autonomous nervous system, pancreatic acinar cells, hepatocytes, Kupffer cells, follicular epithelial cells of the thyroid gland, and macrophages of the lymphatic tissues. Neuronal necrosis, axonal spheroids formation, and astrogliosis were additionally observed in the brain. Ultrastructurally, the cytoplasmic vacuoles consisted of distended lysosomes surrounded by a single-layered membrane. Nonreduced end-rests or sequence of alpha-Man, alpha-Glc, beta(1-4)-GlcNAc, and NeuNAc on lysosomal membrane were revealed by lectin histochemistry. Samples of plants used in the experimental trial contained swainsonine and calystegine and their intermediary derivate. We conclude that I fistulosa induces a glycoprotein storage disease primarily based on the inhibition of the lysosomal alpha-mannosidase by the alkaloid swainsonine.     

A lysosomal storage disease induced by Ipomoea carnea in goats in Mozambique.

A novel plant-induced lysosomal storage disease was observed in goats from a village in Mozambique. Affected animals were ataxic, with head tremors and nystagmus. Because of a lack of suitable feed, the animals consumed an exotic hedge plant growing in the village that was identified as Ipomoea carnea (shrubby morning glory, Convolvulaceae). The toxicosis was reproduced by feeding I. carnea plant material to goats. In acute cases, histologic changes in the brain and spinal cord comprised widespread cytoplasmic vacuolation of neurons and glial cells in association with axonal spheroid formation. Ultrastructurally, cytoplasmic storage vacuoles in neurons were membrane bound and consistent with lysosomes. Cytoplasmic vacuolation was also found in neurons in the submucosal and mesenteric plexuses in the small intestine, in renal tubular epithelial cells, and in macrophage-phagocytic cells in the spleen and lymph nodes in acute cases. Residual alterations in the brain in chronic cases revealed predominantly cerebellar lesions characterized by loss of Purkinje neurons and gliosis of the Purkinje cell layer. Analysis of I. carnea plant material by gas chromatography-mass spectrometry established the presence of the mannosidase inhibitor swainsonine and 2 glycosidase inhibitors, calystegine B2 and calystegine C1, consistent with a plant-induced alpha-mannosidosis in the goats. The described storage disorder is analogous to the lysosomal storage diseases induced by ingestion of locoweeds (Astragalus and Oxytropis) and poison peas (Swainsona).     

Renal pathology in chicks following water deprivation

Total water deprivation of 2-day-old broiler chicks for 24, 48, 72, 96, and 120 hours did not result in visceral urate deposits. Renal tubular changes consisted of increased spaces between membrane infoldings of the distal convoluted tubular epithelium, increased number of cytoplasmic vacuoles in proximal convoluted tubules, and increased mucin production and dilation of collecting ducts.     

Lysosomal storage disease in Sida carpinifolia toxicosis: an induced mannosidosis in horses

REASONS FOR PERFORMING STUDY: This study reports a neurological disease unrecognised until now in ponies in southern Brazil. HYPOTHESIS: Epidemiological data strongly suggests that the ingestion of Sida carpinifolia is involved in the aetiology. We tested the hypothesis that it is an acquired lyosomal storage disease. METHODS: Following the death of 3 ponies, all ponies from the premises were closely monitored; epidemiological data and clinical findings carefully recorded. Fragments of several organs, including CNS, were fixed in neutral formalin and embedded in paraffin-wax. Sections were stained with haematoxylin and eosin. Representative sections of the cerebellum and trigeminal ganglia were submitted to lectin histochemical procedures. RESULTS: The neurological disorder, characterised by stiff gait, muscle tremors, abdominal pain and death, was observed on a farm with 3 hectares of pasture. Three of 11 ponies died 15-20 days after they had been introduced into a new paddock heavily infested by the plant Sida carpinifolia. No significant gross lesions were observed. The main histological findings included multiple cytoplasmatic vacuoles in swollen neurones in the brain, cerebellum, spinal cord, autonomic ganglia (trigeminal and celiac ganglia), and submucosal and myenteric plexus of the intestines. In the kidneys, there was marked vacuolation of the proximal convoluted tubular cells. Sections of cerebellum and trigeminal ganglion were submitted to lectin histochemistry. The vacuoles in different cerebellar and ganglion cells reacted strongly to the following lectins: Concanavalia ensiformis, Triticum vulgaris and succinylated-Triticum vulgaris. CONCLUSIONS: The pattern of staining coincides with that of both swainsonine toxicosis and inherited mannosidosis reports. The histopathological changes were similar to those described in S. carpinifolia spontaneous and experimental poisoning in goats. This disease seems to be similar to Swainsona, Oxytropis and Astragalus toxicosis. POTENTIAL RELEVANCE: S. carpinifolia should be evaluated as a possible cause in the diagnosis of equine neuropathies.     

Mycotoxicosis produced in swine by cultural products of an isolate of Aspergillus ochraceus. I. Clinical observations and pathology.

Pigs fed a ration, 25% of which was rice culture, of Aspergillus ochraceus lost weight or failed to gain and became depressed. Some pigs died and most developed subcutaneous edema, hydrothorax, hydroperitoneum, pulmonary atelectasis, edema of the mesentery and perirenal edema. Microscopic lesions in addition to edema were primarily renal and consisted of tubular degeneration and necrosis, hyaline tubular casts, interstitial fibrosis and tubular cell regeneration. The first change found after 3 days was cytoplasmic vacuolation of the convoluted and straight segments of the proximal tubules. Necrotic proximal tubules were found after 4 days and after 9 days degeneration and necrosis involved predominantly proximal tubular segments. Pigs fed a ration, 12.5% of which was rice culture, for 8 weeks did not develop perirenal edema but had firm kidneys. Extensive interstitial fibrosis of the cortical labyrinth was the principal change. Within the fibrous connective tissue, some tubules were necrotic and others were atrophied.     

Staining pattern of the brush border and detection of cytoplasmic granules in the uriniferous tubules of female DBA/2Cr mouse kidney: comparison among various fixations and stains.

The proximal straight tubules of the female mouse kidney exhibit heavy periodic acid Schiff (PAS) staining in their brush borders and numerous cytoplasmic granules. In the present study, the female DBA/2Cr mouse kidney was examined, using various fixatives (formalin, PFA, PLP, Zamboni's, Bouin, or Carnoy solution) and various staining methods (HE, PAS, alcian blue, periodic acid methenamine-silver (PAM), toluidine blue, azan, or Congo red). Under azan and PAM, the staining pattern of the brush border was similar to that of PAS, and few effects of the fixative were observed. Cytoplasmic granules were clearly detected with PAM as well as PAS. However, these granules were not detected with Carnoy solution. Furthermore, distribution of granules differed between PAS and PAM.     

Ultrastructural alterations in furazolidone-induced cystic testicular degeneration in ducklings

Cystic testicular degeneration was induced in groups of ducklings by feeding furazolidone at 250, 400, 550, or 750 mg per kilogram of feed (ppm) for 4 weeks. In normal ducklings, tall, columnar Sertoli cells predominated in the seminiferous epithelium. In treated ducklings, the mildest ultrastructural alteration was cytoplasmic vacuolation of Sertoli cells. In birds with more severely affected tubules, cytoplasmic vacuolation was more severe and the cells were cuboidal or rounded. The rounded cells desquamated into the tubular lumens, eventually undergoing cytolysis. In birds with the most severely affected tubules, only extensively flattened epithelial cells lined the extremely dilated seminiferous tubules. The interstitium was edematous and compacted between the expanded tubules. Many of the testicular ultrastructural alterations in furazolidone-intoxicated ducklings were similar to those described in the testicles of sodium-intoxicated cockerels.     

Immunochemical Localization of Megalin,Retinol-binding protein and Tamm–Horsfall Glycoprotein in the Kidneys of Dogs

Megalin, retinol-binding protein (RBP) and Tamm–Horsfall glycoprotein (THP) are involved in the renal metabolism of vitamin A in canine species. The presence of megalin, RBP and THP in the kidneys of dogs was investigated using immunohistochemical methods. Megalin was highly expressed in the apical membrane of the proximal convoluted and straight tubule cells. Immunoreactive RBP was detected below the apical plasma membrane, as well as in basolateral granules of the proximal convoluted tubule cells. THP immunoreactivity was seen in the epithelial cells lining the thick ascending limb of the loop of Henle. Furthermore, THP was displayed in a scattered pattern within the distal convoluted tubules. The co-localization of megalin and RBP coincides with biochemical studies that have shown megalin to be responsible for renal RBP absorption in the proximal convoluted tubules after filtration through the renal glomerulus. The presence of THP, the carrier for vitamin A in canine urine, showed that vitamin A excretion in the urine of dogs is not merely a filtration process but also seems to be a pathway located in the distal part of the nephron.     

Mannosidosis in Galloway calves

Mannosidosis was diagnosed in four stillborn Galloway calves and an autolyzed full-term fetus from experimental matings of carrier animals. Gross lesions were moderate internal hydrocephalus, and pallor and enlargement of the liver and kidneys and arthrogryposis. Histologic changes in the central nervous system of each calf were marked foamy vacuolation of the cytoplasm of neurones in the cerebral cortex, thalamus and brainstem, and vacuolation of the Purkinje cells of the cerebellum. Spheroids were common throughout the brain and there was also consistent severe foamy cytoplasmic vacuolation of renal tubular epithelial cells and hepatocytes. The activities of alpha-mannosidase, the lysosomal enzyme whose activity is deficient in mannosidosis, and activities of five other lysosomal enzymes were compared in brain, liver, and kidney tissues of three mannosidosis-affected calves and normal calf tissues. Tissues from the affected calves had a marked deficiency of alpha-mannosidase activity compared with the normal tissues; the greatest deficiency was in the liver (99%) and brain (98%). Activities of the other lysosomal enzymes were elevated in the affected tissues compared with normal. Mannosidosis is a lysosomal storage disease that results from a defect in glycoprotein metabolism and affects man, Angus and Angus-related breeds of cattle, such as Murray greys, and the cat. The congenital disease is caused by an inherited deficiency of the lysosomal enzyme alpha-mannosidase, and is inherited in an autosomal recessive manner. Mannosidosis was recently reported in a number of aborted and stillborn Australian Galloway calves from an experimental breeding trial. This is more detailed account of the histological and biochemical results obtained during the trial.     

Characteristic changes in carbohydrate profile in the kidneys of hereditary nephrotic mice (ICGN strain)

The ICR-derived glomerulonephritis (ICGN) mice consist of heterozygous and homozygous groups and are considered to be a good model for human idiopathic nephrotic syndrome. To reveal changes in cell-surface carbohydrate construction, 24 lectins were applied to kidney sections of 10-, 30- and 50-week-old male heterozygous and homozygous ICGN mice and age-matched male ICR mice. Bandeiraea simplicifolia lectin-I (BSL-I), which specifically binds to alpha-D-galactopyranosyl groups, showed positive staining in the glomeruli of ICGN mice, but not in those of ICR mice. Positive BSL-I staining was observed only in distal tubules of homozygous ICGN mice. Lectin blotting for BSL-I demonstrated characteristic glycoproteins (45, 58 and 64 kD) in ICGN but not in ICR mice, and the levels of these molecules augmented in homozygous ICGN mice with the progression of renal failure. Moreover, succinylated wheat germ agglutinin, Dolichos biflorus agglutinin, Aleuria aurantia lectin and Ulex europaeus agglutinin-I showed positive staining only in the glomeruli of homozygous ICGN mice, but not in those of heterozygous ICGN or ICR mice. The staining intensities of Ricinus communis agglutinin-I, Phaseolus vulgaris agglutinin-E and -L, Lens culinaris agglutinin and Erythrina cristagalli agglutinin (ECL) in the glomeruli of homozygous ICGN mice were stronger than those of heterozygous ICGN and ICR mice. In conclusion, lectin histochemistry provided useful information for the diagnosis and prognosis of nephrotic lesions. Characteristic BSL-I binding glycoproteins may be pathogenic factors which cause renal disease in ICGN mice and are good tools to investigate the molecular mechanism of renal disorders in ICGN mice.     

Renal lesions in rats infected with Rattus serotype hantavirus (SR-11 strain).

Hemorrhagic fever with renal syndrome (HFRS) virus, strain SR-11 (SR) was inoculated intraperitoneally into specific-pathogen-free (SPF) newborn rats, from which the kidney lesions were examined pathologically. The infected rats revealed proteinuria on and after 16 days postinoculation (PI). Histologically, the epithelial cells of the renal tubules showed mild vacuolar and granular degeneration with cytoplasmic inclusion bodies (CIB) on and after 16 days PI. Ultrastructurally, a decrease in number of mitochondria and endocytic vesicles was recognized in the epithelial cells of the proximal renal tubules. Occasionally, both the proximal and distal renal tubular cells had CIB near well-developed Golgi apparatus on and after 13 days PI. Immunohistochemically, CIB were positive for anti-SR nucleocapsid antibody, but negative for anti-SR envelope protein antibody. From the results obtained here, it was concluded that the proteinuria in rats infected with HFRS virus resulted from an insufficiency of reabsorption in the proximal renal tubules, and that CIB consisted of the viral nucleocapsid protein.     

Effect of EDTA on morphology of neutrophils of healthy dogs and dogs with inflammation

Increased basophilia and foamy vacuolation in the cytoplasm of neutrophils in smears made within 1 hour of blood collection suggest inflammatory disease. Due to the mild increase in foamy vacuolation which occurred with time in EDTA, the significance of mildly vacuolated neutrophils in smears prepared a few hours after blood collection is questionable. However, moderate or severe foamy vacuolation should be considered clinically significant. In vitro morphologic changes in neutrophils from healthy dogs in EDTA include clear, discrete vacuoles in the cytoplasm, uneven distribution of cytoplasmic granules, irregular cell membrane and pyknosis. These neutrophils generally lack basophilia, have minimal foamy vacuolation of the cytoplasm and are morphologically different from neutrophils associated with severe inflammation.     

Tubular reabsorption of protein by porcine kidneys during neonatal alimentary proteinuria

The ability of renal proximal tubular cells to reabsorb protein early in postnatal life was investigated using goat haemoglobin as tracer. The haemoglobin was intracardially administered to newborn piglets. The kidneys were fixed for light and electron microscopy 4 h later. Piglets killed immediately after birth and those allowed to suck colostrum for 4 h were used as controls. The proximal tubular cells of newborn, unsuckled piglets already contained absorptive vacuoles. Haemoglobin was absorbed in some absorptive vacuoles of the proximal tubules. The tracer was also demonstrable in the urine, mainly in the form of methaemoglobin. Although proximal tubular cells of newborn piglets are able to absorb protein, this absorption is of limited extent and excess protein is voided with the urine.     

鸡实验性尿酸盐沉积症的肾脏病理学研究

将１２０只３５日龄迪卡商品代蛋用雏鸡随机分为高钙组、高蛋白组和对照组,分别饲喂高钙（含钙３．９％）、高蛋白（含粗蛋白４４．０％）和全价（含钙０．８％,粗蛋白１８．２％）饲料。试验第２５天,各组剖检５只鸡,作肾脏的光镜和电镜观察。结果表明,高钙、高蛋白饲料均能引发尿酸盐沉积。高钙组鸡肾脏肿大,表面及切面有大量尿酸盐结晶;输尿管扩张,管内充满白色尿酸盐沉积物。光镜观察,肾小球肿胀,近曲及远曲小管上皮细胞肿胀、变性。电镜观察,近曲小管上皮绒毛短缩、肿胀、断裂、脱落,线粒体肿胀、嵴断裂;远曲小管线粒体肿胀,呈现空泡,核呈锯齿型。高蛋白组眼观病变不明显,肾脏的光镜、电镜病变与高钙组相似,但损伤程度较轻。作者认为,高钙、高蛋白所致肾脏损害是鸡尿酸盐沉积症的重要原因。     

Recombinant rabies virus vaccine strain SAD-L16 inoculated intracerebrally in young mice produces a severe encephalitis with extensive neuronal apoptosis

Seven-day-old ICR mice were infected by intracerebral inoculation with recombinant rabies virus vaccine strain SAD-L16. Infected mice developed severe and fatal encephalitis with rabies virus-infected neurons in widespread regions of the brain. There was extensive neuronal death with predominant features of apoptosis, as assessed by light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, and immunohistochemical staining for activated caspase-3. Although SAD-L16 is a neuroattenuated rabies virus, it is fully capable of spreading efficiently and inducing widespread neuronal apoptosis in the immature mouse brain.     

Decreased expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in the kidneys of hereditary nephrotic (ICGN) mice

Matrix metalloporoteinases (MMPs), which are dominantly regulated by tissue inhibitors of metalloproteinase (TIMPs), play important roles in extracellular matrix (ECM) degradation and are involved in the progression of kidney diseases. In glomeruli and tubulointerstitum of hereditary nephrotic (ICR-derived glomerulonephritis: ICGN) mouse kidneys, hyper-accumulation of ECM components occurred, and MMP activity decreased. In the present study, because lower levels of MMP activity may contribute to the progression of renal fibrosis in ICGN mice, Western blotting analysis and immunohistochemical staining for MMPs and TIMPs were performed to verify the expression levels of these proteins. Levels of MMP-2, MMP-9, MT1-MMP, TIMP-1 and TIMP-2 in the kidneys were decreased in ICGN mice in comparison with normal ICR mice. These results indicate that small amounts and low levels of activity of MMPs cause the progression of renal fibrosis in ICGN mice.